Phosphorylation and dephosphorylation of proteins functions as a dynamic mechanism at checkpoints that starts and stops cell growth. This process is critical for normal development and differentiation. The balance involves the opposing reactions of cyclin-dependent protein kinases (CDK) and protein Ser/Thr phosphatase type-1 (PP1). The CDKs are regulated by periodic synthesis and degradation of the cyclin subunits, by phosphorylation of their catalytic subunits, and by binding of various inducible inhibitor proteins. The tumor suppressor p53 transactives expression of CDK inhibitors, and failure to produce these inhibitor proteins results in cell transformation and tumor formation. The goal of this project is to understand unique mechanisms that regulate the Inh2. This will be done by 1) determining the phosphorylation of Inh2 in different phases of the cell cycle by metabolic labeling of cells and phosphopeptide mapping, 2) testing the effects of mutations in Ser phosphorylation sites in Inh2 on nuclear export and import on Inh2 and 3) mutating PEST regions of and Ser phosphorylation sites to alter proteolysis of Inh2. An Inh2-green fluorescent protein (GFP) fusion protein that shows cell cycle dependent redistribution between cytosol and nucleus will be mutated to show which Ser sites are required for Inh2 distribution and degradation. Epitope tagged Inh2 mutants expressed in bacteria and microinjected into fibroblasts provides a complementary approach. Tagged Inh2 will be used to isolate Inh2 complexes to identify components associated with Inh2 at different times in the growth cycle. Together, these studies will elucidate new roles for Inh2 and PP1 in growth control, and expose the potential of defects in these proteins in human cancers.